Mortierella alpina is an important arachidonic acid (ARA) industrial production fungus. The produced polyunsaturated fatty acids (PUFAs) have a reasonable composition that contains high level of ARA, which have a record of complete safe for applications in food. By far, the studies on M. alpina were mainly focused on the strain breeding and the optimization of fermentation conditions. The gene transformation system of M. alpina has not been well established. This is a great obstacle to the studies on the mechanism of fatty acid synthesis and metabolic engineering of M. alpina. Auxotrophic marker, antibiotic resistance marker and fluorescent reporter gene are three well-used selective marker for gene transformation in filamentous fungi. The auxotrophic is applicable for industrial production, because there is no residual exogenous resistance gene. Therefore, the auxotrophic strains are important for industrial breeding microorganisms, genetics, medicine, food and biotechnology engineering. Currently, the auxotrophic strains of filamentous fungi are mainly generated by the mutation method, which is inefficient and often causes random unknown mutations in the genome DNA sequences. These unknown mutations may bring unpredictable problems for the future genetically engineering and industrial production.
Constructing auxotrophic through homologous recombination can knock out the target gene without affecting the function of the other genes. Compared to random mutations, homologous recombination is more efficient and repeatable. Therefore, directly interrupt the target gene via homologous recombination is an optional way in generating auxotrophic strains. In filamentous fungi, homologous recombination is affected by many factors: the length, similarity, G/C percentage, transcription of target gene, non-homologous end joining and chromatin structure, as well as the transformation method. In some yeast, homologous recombination could be achieved with a relative short homologous DNA sequence of 50 bp to 100 bp. Whereas in filamentous fungi, homologous DNA sequence often needs to be over 1K bp even longer. The homologous recombination probability may differ a lot among strains and genes, which may strongly affect the experiment. Orotate phosphoribosyltransferase (OPRTase) is a key enzyme during uracil metabolic in M. alpina. The M. alpina auxotroph could be generated by inactivation the OPRTase coding gene ura5. However, ura5 gene has an extremely important role in the cellular processes of life, resulting in very sensitive self-defense and repair mechanisms of the role of eukaryotic cells. Construction of ura5 uracil auxotrophic strain using gene knockout method in filamentous fungi is seldom publicly reported.
The gene manipulation system of filamentous fungus has not been well established, mainly because it is difficult to be transformed. Agrobacterium tumefaciens-mediated transformation (ATMT) method has been gradually applied in filamentous fungi, which have four advantages compared to other transformation methods. First, the recipient could be spores or mycelia without preparing protoplasts. Second, the mononuclear spores as a recipient can avoid transformants instability caused by multicore mycelium. Third, the method uses a natural transformation vector system having high conversion efficiency and high success rate. The plasmid can hold large fragments of DNA with a single copy insertion into genome. Fourth, a relative higher homologous recombination rate can be achieved.
The M. alpina uracil auxotrophic strain is the prerequisites of the gene manipulation of this important industrial PUFA production fungus. This uracil auxotrophic strain could be applied in both theoretical research of fatty acid synthesis and accumulation and genetically engineering to breeding super PUFA production industrial strain.